In 1907, Alois Alzheimer described the case of a 51-year-old woman with a rapidly degenerating memory who, after a swift deterioration, died severely demented four years later. This condition, which now bears Alzheimer's name, describes a fatal degenerative dementing disorder with initial mild memory impairment that progresses unrelentingly to a total debilitating loss of mental and physical faculties. Following symptom onset, the course of the disease varies considerably from a few years to over 20 years, with a mean survival of approximately 8 years. M. A. Smith, “Alzheimer Disease,” Internat. Rev. Neurobiol. 42:1 (1998).
Alzheimer disease affects 10-15% of individuals over 65 years and up to 47% of individuals over the age of 80. In both clinical and autopsy series in the United States and Europe, Alzheimer disease accounts for approximately two-thirds of all dementias affecting elderly individuals. D. A. Evans et al., J. Am. Med. Assoc. 262: 2551 (1989).
The most common and distinctive lesions present with the diseased brain are the neuritic senile plaques and neurofibrillary tangles. The major protein component of senile plaque cores and vascular amyloid is a small polypeptide of approximately 4.2 kDa termed amyloid-β. A significant fraction of this protein is found to be associated with the cytoskeleton, presumably through its interaction with the microtubule-associated τ (“tau”) protein. It is believed that the increased phosphorylated status of tau protein represents one of the earliest neuronal changes prior to the development of neurofibrillary tangles.
Unfortunately, because of the heterogeneity of the factors thought to be responsible for Alzheimer disease and the lack of an animal model displaying the full spectrum of pathological changes, successful pharmacological interventions have not been established. What is needed is an easy, reliable method to determine the safety and efficacy of candidate therapeutics for the treatment and/or prevention of Alzheimer disease.
Definitions
The term “drug” as used herein, refers to any medicinal substance used in humans or other animals. Encompassed within this definition are compound analogs, naturally occurring, synthetic and recombinant pharmaceuticals, hormones, neurotransmitters, etc. The present invention contemplates screening test compounds to identify a useful drug for the treatment of Alzheimers.
Most current attempts at therapeutics for Alzheimer disease are directed at neurotransmitter deficiencies. The term “neurotransmitter” includes any compound which functions in the nervous system to result in the transmission of chemical signals between cells. Examples of neurotransmitters include, but are not limited to neuropeptides, acetocholine, and amino acids (e.g., GABA). Other compounds are also contemplated, including dopamine, norepinephrine, etc.
The term “GABA” refers to γ-aminobutyric acid, a major inhibitory neurotransmitter in both vertebrates and invertebrates. Kuffler and Edwards, J. Neurophysiol., 21:589 (1965; Otuska et al., Proc. Natl. Acad. Sci USA 56:1110 (1966); Usherwood and Grundfest, J. Neurophysiol., 28:497 (1965). The term “GABA receptors” thus refers to structures expressed by cells and which recognize GABA.
The present invention contemplates the detection of a variety of therapeutic compounds, including but not limited to compounds that inhibit re-entry of neuronal cells into the cell cycle. Such compounds may be agonists or antagonists.
The term “agonist” refers to molecules or compounds which mimic the action of a “native” or “natural” compound. Agonists may be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may be recognized by receptors expressed on cell surfaces. This recognition may result in physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present.
The term “antagonist” refers to molecules or compounds which inhibit the action of a “native” or “natural” compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors that are recognized by an agonist. Antagonists may have allosteric effects which prevent the action of an agonist. Or, antagonists may prevent the function of the agonist.
The term “host cell” or “cell” refers to any cell which is used in any of the screening assays of the present invention. The present invention contemplates “host cells” or “cells” in their natural states as well as genetically altered cells.
As used in the present invention, the term “transformation” refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. “Transformation” includes but is not limited to such methods as transfection, microinjection, electroporation, and lipofection (liposome-mediated gene transfer). Transformation may be accomplished through use of any expression vector.
The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a fill length coding sequence or by any portion of the coding sequence so long as the desired activity is retained. In the case of the present invention, oncogenes are contemplated and the desired activity is to cause neuronal cells and tissue to exhibit characteristics associated with re-entry into the cell cycle. Such characteristics may be cellular antigen expression (e.g. phospho-tau expression). On the other hand, a simple characteristic that can be readily measured is the incorporation of a nucleic acid precursor (indicating that the cells are traversing the S-phase of the cell cycle). Such precursors include, but are not limited to, 8-bromodeoxyuridine and tritiated-thymidine.
The term “wild-type” refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified” or “mutant” refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The present invention contemplates wild-type oncogenes (e.g. from tumors) as well as oncogenes generated by mutation.
The term “oligonucleotide” as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends.